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Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide
Induction of Primary and Inhibition of Secondary Antibody Response to Hapten by Hapten Conjugates of Type Ill Pneumococcal Polysaccharide’ S.
P.
LERMAN,
”
T. J.
KOMANO,
G. J. Department
of Pathology, New
New Fork,
a
J. J.
FROND,
&I.
HEIDELBERGER,
AND
THORBECXE York New
Unizlevsity School York 10016
of Medicble,
Trior dinitrophenylated pneumococcal polysaccharide type III (TNPor DNPSIII) ) induced a primary 19s anti-TNP response without generating immunological memory to the hapten in LAF, mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. a6ortus organisms (DNP-BA) induced both 19s and 7s primary responses and memory to the hapten. Spleen cells from mice immunized with TNPKLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLHprimed spleen cells for as short as 5 min at 0°C with 10 pg of TNP-SIII per milliliter virtually abolished their ability to give 19s and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.
INTRODUCTION Immunization with hapten coupled to a polysaccharide carrier which, by itself, induces thymus-independent immune responses, may give rise to anti-hapten antibody (1, 2). The mechanism of cellular interaction involved, if any, is unclear, but it has been suggested that direct mitogenic activity of these carriers for B cells is of prime importance in inducing T-cell independent responses (3, 4). The anti-hapten response may have similar properties as the response to its carrier in that it is limited to antibody production of the 19s class (1, 2) and deficient in the formation of immunological memory (5). On the other hand, hapten conjugates of nonimmunogenic molecules such as a copolymer of u-glutamic acid and D-lysine ( D-GL) (68)) syngeneic y-globulin (9, lo), and syngeneic red cells 1 Supported by Grant AI-3076 from the United States Public Grant QFB13592 AI from the National Science Foundation. 2 Supported by USPHS Training Grant No. GM000127. 3 Recipient of National Institutes of Health Training Grant National Institute of General Medical Science. 321 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Health
No.
Service
5GM01668-11
and
in part
by
from
the
322
LERMAN
ET
AL.
not appear to induce immm~e responses, but on the contrary may induce tolcrante to the hapten (11). It has been suggested that “no11iilllllllnoge~lic” hapten-carrier conjugates might have important applications in the abrogation of ongoing but mnwanted humoral responses (12). It would be of interest if hapten conjugates could be found which specifically abolish some but not all facets of the immune response to the hapten. For example, B-cell tolerogens such as the dinitrophenylated (DNP) D-GL inhibit antibody production while leaving intact T-cell functions with specificity for the hapten such as delayed and contact hypersensitivity (13, 14). Another promising approach to this problem was initiated by Mitchell et al. (15), who demonstrated interference with the 7s antibody memory response to DNP-hemocyanin through injection of the DNP-lysine conjugates of pneumococcal polysaccharide type III (DNP-SIII) . The present studies were undertaken to determine (i) whether the trinitrophenyl-lysine conjugates of SIII (TNP-SIII) could induce an immune response to TNP with generation of immunological memory, and (ii) whether or not the inhibition of 7S memory expression observed by Mitchell et al. (15) also encompassed 19s memory and could be obtained by in vitro incubation of memory cells.
do
MATERIALS
AND
METHODS
Animals and Immunizations Male young adult LAFl mice (Jackson Labs, Bar Harbor, ME) were used throughout. Donor mice were immunized by intravenous injection of 0.3 mg TNPla-KLH usually with 10 pg E. c&i endotoxin (Difco Laboratories, Inc., Detroit, MI). In a few experiments donor mice received 0.1 ml of a 0.3% (v/v) suspension (equivalent to 2 X 10R) of killed Brucelh abortus (BA) organisms. Whenever the interval prior to sacrifice of donor mice exceeded 2-4 months, they received a second dose intraperitoneally. Donors were used no earlier than 1 month after their last priming injection. Immunizations with TNP and DNP conjugates of SIII or of BA were always by intravenous injection of antigen in dosages as described in the tables. On the day prior to transfer, recipient mice received 630-750 R 13iCs v-irradiation from a gammator M (Radiation Machinery Corp., Parsippany, NJ ) . Antigens DNPSO-SIII and TNP,,,-SIII were prepared by conjugation to SIII of r-DNPL-lysine HCl (Sigma Chemical Co., St. Louis, MO) or e-TNP-L-lysine HCl (Nutritional Biochemicals Corp., Cleveland, OH), respectively, by the cyanogen halide method of Axen and Ernback (16) as described by Mitchell et al. (15). Trinitrophenylated sheep erythrocytes (TNP-SE) were prepared according to the method of Rittenberg and Pratt (17). TNP-BA and DNP-BA were prepared by the method of Little and Eisen (18). Briefly, trinitrobenzene-sulfonic acid (TNBS) was recrystallized from the commercial preparation (Eastman Kodak Co., Rochester, NY) by cooling from hot 1.0 N HCl and washing with cold HCl. To 20 mg of recrystallized TNBS or of dinitrobenzene-sulfonic acid (Eastman Kodak Co., Rochester, NY) dissolved in 2.0 ml of a 2% KzC03 solution were added 2.0 ml of packed BA (U.S. Department of Agriculture) with stirring. Stir-
BLOCK
OF
IZNTI-ITAPTEN
MEMORY
lo2o-7
RY
11 .‘iPTEN-SIII
323
6 Days
After
Injection
FIG. 1. Primary anti-TNP responses of LAF, mice expressed as PFC/spleen at various days and TNP-B.4 (A-A) or 1 ,~g (O-0) and after injection of DNP-BA (n---A) 100 pg (O--O ) DNP-SIII. Each point represents the mean f SE for 2-9 mice. Shaded area denotes the mean PFC level for normal spleen 5 SE measured with TNP-sheep erythrocytes. The numbers of PFC/spleen against sheep erythrocytes were subtracted from all values.
ring was continued for an additional 2 hr at room temperature and overnight at 0°C. The mixture was dialyzed successively against distilled water, 0.1 M KC2H302, and finally 0.15 &f NaCl. Dinitrophenyl groups were conjugated to human gamma globulin (HGG) and keyhole limpet hemocyanin ( KLH) (Mann Laboratories, Orangeburg, NY) (18). The extent of coupling of DNP to HGG, KLH, and SIII was estimated from the molar extinction coefficient, 17,400, at 360 nm with 100,000 as the unit of molecular weight for the protein and polysaccharide carriers (15). The degree of TNP conjugation was estimated using the molar extinction coefficient, 15,400, at 346 nm. SIII was prepared as described previously (19), and the amount of polysaccharide present in each of our hapten-polysaccharide conjugates was determined by the phenol-sulfuric acid reaction of Dubois et a.1. (20). Cell Transfers
The spleens of donor mice were teased into a cell suspension in Medium 199 (Microbiological Associates Inc., Bethesda, MD) and washed twice. Cells were either injected intravenously into irradiated recipients with antigen or were incubated with antigen in zitro prior to transfer as described under Results. Assays
Numbers of antibody-forming cells per spleen to TNP were enumerated by localized hemolysis in gel of TNP-SE (17). A s a control, spleen cell suspensions were also plated against uncoated SE and the values obtained subtracted from those for PFC against TNP-SE. Rabbit anti-mouse Ig was used for development of indirect PFC (21). The numbers of 7S PFC were calculated by subtracting the direct PFC from the indirect PFC. Sera from recipient mice were titrated for anti-TNP and anti-KLH by passive hemagglutination. Indicator cells were tanned cells (22) to which had been adsorbed 0.05 mg TNP1.7-ovalbumin or KLH per milliliter of 2.5% tanned SE.
324
LERMAN
ET
i\L.
RESULTS Primary
Response
to Hapten
Presented
011 the BA and SIII
Carriers
Initial studies of responses to a single injection of DNl’-]:A, TNP-BA, DNl’SIII, and TNP-ST11 showed that all induced a rise in the number of PFC per spleen with anti-TNP specificity, but with large differences in effectiveness between the SIII and the BA carrier. Figure 1 shows the responses to DNP-BA and TNP-BA. Peak PFC/spleen values were reached on day 3, and the number of antibody-forming cells remained high for several days. TNP-BA always gave a much higher response than three different preparations of DNP-BA, and at the dose used was also more effective than either TNP-KLH or DNP-KLH given as a single intravenous dose of 100 pg. The mean PFC/spleen on day S after TNP-KLH was 4175 as compared to 583 for DNP-KLH. 4 The high immunogenicity of BA conjugates might be due to their probable content of endotoxin. Indeed, immunization with DNP-KLH, given together with 50 pg of E. coli endotoxin, resulted in a response at least as high as that to DNP-BA. Although the majority of the PFC detected through day 6 after injection of KLH or BA conjugates were 19S-producing, a significant percentage of animals also showed 7S antibody-producing cells on day 6 of the primary response. The response to DNPor TNP-SIII, on the contrary, did not show a significant 7s component at any time. Peak PFC/spleen values after an optimal (1 pg) dose of DNP-SIII were reached on day 3 and declined thereafter (Fig. 1). Ten micrograms of DNP-SIII induced a similar response, but that to 100 pg was much lower (Fig. 1) . Control LAFl mice injected with 0.1 ml of 10m5or of 1c6 M TNP-lysine containing 1 pg unconjugated SIII did not show any splenic PFC above background 4 days later. The response to TNP-SIII was slightly higher than to DNP-SIII but otherwise was similar. Mean PFC/spleen values on days 4-5 were 3550 after 1 pg, 3218 after 10 pg, and 724 after 100 pg of TNP-SIII. Experiments in which immunization with DNP-SIII was attempted 1 week after a previous tolerogenic or immunogenic dose of SIII were difficult to interpret, since both dose levels of SIII used appeared to lower somewhat the subsequent response to DNP-SIII. Secondary
Response
in Yivo
to Haptens
Presented
on BA
and SIII
Carriers
Mice reinjected with 1 pg DNP-SIII 3 weeks after a primary injection of 1, 10, or 100 pg DNP-SIII did not show a secondary response. Direct PFC/spleen values measured 6 days later were no different from those in unchallenged mice and were similar to those seen 5 days after a primary injection (Expt 1, Table 1). 7s PFC were absent. DNP-BA induced a slightly higher response comparable to the one seen on day 6 after a primary injection of this antigen. There was no 4In this experiment all PFC determinations were made against TNP-SE rather than DNP-SE. That higher PFC values after immunization with TNP as compared to DNP conjugates were not due to the method of detection used was shown in unpublished observations with Dr. E. Goidl and Dr. G. W. Siskind, Department of Medicine, Cornell University Medical Center, New York, in which PFC detected with DNP-SE as indicator cells were also more numerous after immunization with TNP conjugates. Thus, TNP conjugates appear to have higher immunogenicity for LAFl mice than do DNP conjugates.
BLOCK
OF
ANTI-IIAPTEN
MEMORY
BY
IIAl’TEN-SIII
~MhlliNOl.OGlCAI,
h!EMOR\'
TABLE INABILITY
OF UNP-SIII
TO Imuce
1
Challengmg antigen
Priming antigen
Mean
Expt
Direct (1 ,ug)
Expt
1”
3.25
f 0.09 (1760)
3.39
f 0.15 (2425)
DNP-BA
3.52
z!c 0.10 (3290)
3.53
f 0.17 (3405)
Direct
3.35
TNP-BA
DNP-SIII
(10 fig)
3.18~ (1510) 3.36
f 0.07 (2270)
3.19
i 0.13 (155.5)
DNP-BA
3.66
f 0.10 (4535)
3.67
zk 0.12 (4670)
TNP-BA
DNP-SIII
(100
rg)
DNP-BA
A 0.13 (2255)
3.36 f 0.16 (2270)
4.45 f 0.07 (28,150)
4.65 f 0.13 (44,500)
3.30
3.29
f 0.12 (2010)
4.38 =!z 0.25 (24,200) 3.18~ (1520) 3.19
f 0.12 (1555)
3.23
i 0.08 (1710)
DNP-BA
3.45
f 0.07 (2785)
3.46
zk 0.06 (2850)
3.1Sc (1510) 3.58
41 0.06 (3830)
3.64
DNP-BA
3.69
f 0.08 (4930)
4.06 zt 0.09 (11,450)
3.14c (1370)
4.S6 f 0.22 (36.480)
3.16< (1460)
DNP-SIII
None
zk 0.07 (1940)
3.20c (1.570)
DNP-SIII
None
Indirect
3.11c (1300)
DNP-SIII
None
2”
__~
Indirect
DNP-SIIl*
None
TO DNP
log PFC/spleen & SE (geometric mean)
.___..~
DNP-SIII
32.5
zt 0.06 (4410’
3.3.V (2230)
a Interval between injections was 21 days (Expt 1) or 14 days each group varied from 4 (Expt 1) to 2 or 3 (Expt 2). Determination day 6 (Expt 1) or day 5 (Expt 2) after challenge. b Challenge dose was 1 pg DNP-SIII in all experiments. c Recipients’ spleens in these groups were pooled prior to assay
(Expt 2). Numbers of PFC/spleen
for
of mice in was done on
PFC.
tolerance to DNP in mice previously injected with 100 pg DNP-SIII since they were still able to respond to DNP-BA, although possibly somewhat less than normal mice. In Expt 2, mice reinjected with DNP-BA and TNP-BA 2 weeks after priming with DNP-911 gave similar results. Responses comparable to those
326
LERMAN
ET
TABLE
Donors primed with
AL.
2
Recipients challenged with
-VI/lean log PFC/spIeen (geometric mean)
2.65
9’
Expt
Indirect
Direct DNP-BA
AI 0.05
3.68
(442) DNP-SIII”
1.76
f
0.45
1.81
Direct
f 0.09 (4810) f
0.18
DNP-KLH*
DNP-SIII
(1 ,.‘g)
(100 pg)
2.36
f 0.16 (229)
2.26
f
0.08
(182)
DNP-BA
3.28
zk 0.13 (1905)
3.21
f 0.06 (1620)
DNP-SIII
3.19
f 0.19 (1550)
3.05
zt 0.09 (1130)
DNP-BA DNP-SIII None
2.75
3.2@ (1840) 3.22
f 0.08 (1660)
2.85
f 0.21 (709)
3.08c (1200)
Indirect
zt 0.04 (1290)
3.70
rt 0.14
2.68
f 0.13 (1160)
.-.
@ -___
(563) 3.07
None
DNP-SIII
3.11
(65)
(57)
None
SE ____-.
Expt
DNP-BA
f
f 0.08 (5200) f
0.18
(479) 3.11
f 0.14 (1305)
2.93c
2.94c
(851)
(870)
3.08c (1190) 2.87
f
0.18
(742) 2.79
f 0.14 (617) ND”
a Interval between priming and transfer was 16 weeks for the DNP-BA-primed spleen cells and 4 weeks: for DNP-SIII-primed cells in Expt 5, and 3 weeks for DNP-BA-primed spleen cells in Expt 6. Irradiated recipients received 3-3.5 X lo7 cells (Expt 5) or 2 X lo7 cells (Expt 6) together with the challenging antigen. Receipients’ spleens were assayed for PFC 7 days later (4-5 mice per group). * All challenges with DNP-SIII employed 1 rg DNP-SIII except for Expt 5 (donors primed with DNP-BA), where the dose was 50 pg DNP-SIII. Dose of DNP-KLH was 100 rg. c Recipients’ spleens in these groups were pooled prior to assay for PFC. * Not determined.
after a primary injection of these antigens were found on day 5 after challenge, and a 7s component was detectable as also seen after the primary injection. In contrast, mice primed with DNP-BA gave a secondary 7s anamnestic response to DNP which was not demonstrable after challenge with DNP-SIII. There was a slightly higher 19s response to DNP-SIII in these mice than in those primed with DNP-SIII. Adoptive
Secondary
Responses
to DNP-BA
and DNP-SIII
Since it was conceivable that the presence of antibody to the SIII carrier molecule in the sera of primed mice was inhibiting the secondary response to
BLOCK
OF
ANTI-HAPTEN
MEMORY ‘TARI,E
~\RII.ITY
OF DW-I%\
Recipients challenged
AKD
BY
3
DNP-HGG TO ELICIT AN XDOPTIVE DNP-KLH-PKIMED SPI.EIX CELLS hlean
with
327
HAPTEN-SIII
Smmna~r
RESPONSE
log PFC/spleen =t SE (geometric mean) Expt
Expt 31’ Direct Direct
+ Indirect
DNPzG-KLH*
3.21
f 0.08 (1623)
3.38
f 0.09 (2380)
4.40 * 0.04 (25,200)
DNP-RA
3.12
f 0.04 (1320)
3.26
f 0.09 (1830)
3.81
f 0.22 (6460)
DNPj-HGG
2.92
0.05
3.28
f 0.04 (1895)
3.70
f 0.04 (5020)
0.43
3.01
f 0.21 (1025)
3.20
f 0.08 (1585)
f (832)
DNPtg-HGG
1.26
f
(18) n Spleen cells were taken 2 weeks (Expt 3) or 3 KLH with or without 10 rg E. coli endotoxin. 2 X 107 (Expt 4) spleen cells together with the assayed for PFC 6 days (Expt 3) or 7 days (Expt b Challenge dose of antigen, 0.2 mg DNP-KLH
FKOY
weeks {(Expt 4) after injection of 1 mg DNPZBIrradiated recipients received 10’ (Expt 3) or challenging antigens. Recipients’ spleens were 4) later (4-5 mice per group). or 0.1 mg DNP-HGG.
these conjugates, adoptive secondary responses after transfer of 2-3 x lo7 spleen cells from immunized mice were studied. Table 2 shows the results of these experiments. Cells taken 4 weeks after injection of DNP-SIII did not respond to either DNP-BA or DNP-SIII, giving similar PFC values in recipient spleens with or without antigenic challenge at the time of transfer. DNP-BA-primed spleen cells showed definite 7s and weak 19s anamnestic responses to DNP-BA but none to DNP-SIII. These data implied that DNP-SIII was unable to generate memory cells to DNP and, thus, behaved like SIII alone, which is known to induce 19s responses without generating detectable memory (22). However, the possibility existed that DNP-SIII had generated memory cells but was unable to challenge them and that DNP-BA could not challenge this memory to DNP-SIII because of the difference in carrier. Although BA itself is a largely thymus-independent antigen with respect to challenge of memory cells (23), the DNP-BA conjugate might not be equally thymus independent. We therefore tested the capacity of DNP-BA to challenge memory cells generated by DNP-KLH. Spleen cells from DNP-KLHprimed mice were taken 2 weeks (Expt 3, Table 3) or 3 weeks (Expt 4, Table 3) after immunization and challenged up011 transfer (2 x 10’ cells/mouse) with DNPKLH, DNP-BA, or DNP-HGG conjugates. DNP-BA was much more efficient than DNP-HGG in challenging these memory cells, although it induced a lower 7S response than the homologous DNP-KLH conjugate. It should, therefore, have been capable of challenging memory cells to DNP-SIII if such cells had been present.
328
LERMAN
ET
AL.
The previous experiments showed that DNP-SIII neither generated nor challenged DNP memory cells. We next examined the ability of these conjugates to b10clc the responsiveness of memory cells in V~ZIO.Table 4 shows that, when haptenHA- or hapten-KLH-prinie~l memory cells were transferred with the homologous antigen and varying doses of the hapten-SIII conjugates, both 19s and 7S nienlory cells did not respond to the homologous conjugate. Even as little as 0.5-l pg of hapten-SIII resulted in significant inhibition of both responses. In two of the experiments in which this comparison could be made the 19s responses appeared slightly less sensitive to inhibition than the 7s responses (Expts 11 and 12, Table 4). When passive hemagglutination titers to TNP of the recipients’ sera were compared with values for PFC/spleen, agreement between these two measures of immune responsiveness of the transferred cells was noted. In experiments in which the PFC/spleen values indicated an effective block of the response, the anti-TNP titers of the sera were also greatly reduced, while a less effective block of the PFC/spleen response resulted in a smaller reduction in titer. For example, in three typical experiments, the control recipient groups had mean log2 serum titers ranging from 10.0 to 10.8, the groups which did not receive antigen had mean log, titers of 2.0-2.7, and the mice receiving TNP-KLH with 5-50 pg TNP-SIII had mean log2 titers of 2.3-3.3. Mice receiving a lower dose of the blocking antigen (0.5-l pg) had titers of 7.4-8.7. Further studies showed that a simple preincubation with hapten-SIII for as short as 5 min at O”C, followed by washing of the cells and transfer into irradiated recipients together with the homologous conjugate, was sufficient to block partially the response of TNP memory cells (Table 5). Excellent correlation was again found between results obtained for PFC per spleen and for anti-TNP titers in recipients’ sera (Table 6). Incubation at 0°C was no less effective than at 37”C, and prolonging the incubation to 60 min did not increase the degree of inhibition (Table 5)) nor did increasing the concentration of DSP-SIII to 200 rg/ml (45% vs 417*). Although the results were somewhat variable, TKP-SIII was more effective than DNP-SIII, particularly at the 10 pg/ml level, possibly because of the much higher degree of conjugation of ST11 with TNP than with DNP. UThile only one experiment was included in Table 5, good inhibition was obtained at this level of TNP-SIII in several additional experiments. It was noted that both 19s and 7S memory cells were sensitive to the blocking effect of preincubation. Preincubation with 40 pg DNP-SIII/ml, followed by washing, was slightly less effective in blocking the subsequent response to TNP-KLH than the simultaneous injection of 0.5 PLgof DNP-SIII (Expt 12, Tables 4 and 5). Additional experiments (not in the tables) showed that incubation of TNPKLH primed cells with 100 pg/ml of either DNPB-HGG or DNPhS-HGG, followed by washing had no detectable blocking effect on the subsequent adoptive response to TNP-KLH. Controls
for Specificity
of the phenomenon was established by an experiment in which cells to burro erythrocytes were exposed for 1 hr at 0°C to 40 pg TNP-SIII/ml.
Specificity primed
and Suppressor Eflects
S-50 0.5 1 l-50 None
+ + -
100 (1660) 4 20 10 1
19s
Expt
8 7s
ANTIGEN
100 (1790) 3 37
TO HOMOLOGOUS
4
7s
9=
100 (1339) 3
Expt
REMAINING
TABLE
7s
10~ 19s
in Expts were
and antigen
16
53
Expt
16
100 (14,904)
7s
not
significantly
above
Expt
7s
for
(0.1
2
4
100 (35,329)
12
the values
TNP-KLH
19
41
100 (2891)
19s
12 it was
ANTIGEN
9, 11, and
11
OF BLOCKING
100 (16,932)
IXJECTION
(PFC/spleen)
100 (1501)
Expt
7.
AFTER
in Expts 7 and 10 was DNP-BA, in Expt 8 it was TNP-BA, lo-12 was DNP-SIII, and in Expts 8 and 9 it was TNP-SIII. are not included because the 19s responses to homologous
3
(6371
100
Expt 7~ 7s
OF RESPONSE
a The homologous challenging antigen * The blocking antigen in Expts 7 and c In these experiments the 19s values injected without antigen.
None
Blocking antigen6 kd
+
Homologous antigen challenge5
PERCENT
cells
mg).
I I (40’
(10) (40) (10-20) onlyb
TNP-SI
TNP-SIII DNP-SIII DNP-SIII Medium
60 5 5 60 60 60
60
Incubation time (min)
34 16
7s
83 <1
100 (14,904)
11
RESPOXSE
Expt
100 (16,932)
19s
OF SECONDARY
Expt
19
28
100 (2891)
19s
TO TNP-KLH
7S
2
9 18
100 (3289) 75 23
19s
Expt
5
100 (26,389) 23 8
7S
13
‘% (PFC/spleen)”
INCUBATION
11
100 (3529) 3.5
19s
Expt
OF PRIMED
or TNP-SIII at O’C as indicated. They were determined 7 days after transfer.
100 (35,329)
12
REMAININGAFTER
D Cells were preincubated at 2 X 10’ cells/ml with or without DNPcells per recipient with 0.1 mg TNP-KLH. PFC/spleen in recipients b Control cells, not challenged with TNP-KLH after transfer.
only
incubated with CudmU
Medium
Cells
PERCENT
TABLE
75
were
19s
twice
3
12
Expt
DNP-
100 (9671)
WITH
washed
100 (28,321) 3
14
CELLS
7S
prior
19s
40 52 4 of l-2
Expt
100 (1356)
to injection
18
100 (13,922)
15
ORTNP-SIII
7s
X lo7
61 81
100 (1902)
16
F
3
2i
E
E
BLOCK
INHIBITORY
EFFECT
OF
Expt
Medium
only
Medium
Geometric mean PFC/spleen
TNP-KLH Medium
SPLEEN
ANTI-TNP
RUT
KOT
ON
CELLS Expt
14
17
~ Mean logz hemagglutination titer
31,850
TNP-KLH only
331
HAPTEN-SIII
ON ADOPTIVE
OF TNP-KLH-PRIMED
antiTNP
TNP-SIII
BY
WITH
RESPONSES
Cells injected with
MEMORY
TABLE 6 TNP-SIII
OF PKEINCUBATION
ANTI-KLH Cells incubated witha
ANTI-HAPTEN
only
10.8
1,514 606
a Cells were preincubated at 2 X lo7 cells/ml at 0°C. They were washed twice prior to injection per recipient with 0.1 mg TNP-KLH. PFC/spleen mined 7 days after transfer.
2.8 2.7
Geometric mean PFC/spleen
antiKLH 13.3
14.3 7.0
Mean log, hemagglutination titer antiTNP
25,365 4,562 6i7
9.6 4.8 4.0
antiKLH
15.6 7.0 17.7
with or without 40 pg/ml TNP-SIII for 60 min of 1 X 107 (Expt 14) or 2 X lo7 (Expt 17) cells vs TNP and serum antibody titers were deter-
The adoptive memory response of these cells, approximately 6000 7s PFC/spleen, was totally unaffected. Furthermore, while serum titers to TNP of recipients were reduced in parallel with numbers of PFC per spleen, the preincubation of donor cells with the blocking antigen had no detectable effect on the response to KLH, the carrier protein, as determined by serum antibody titers (Table 6). Also, transfer of a combination of unincubated TNP-KLH memory cells with an equal number of such cells after incubation with TNP-SIII and washing gave similar responses to those of unincubated cells showing both the absence of suppressor activity and of excess TNP-911 with the incubated cells. DISCUSSION The results indicate that DNP-SIII, like DNP-levan (2), induces a primary response to DNP in mice which is dose dependent in the sense that high doses (100 pg) give a lower response than low doses (0.5 pg). The response to the hapten on the SIII molecule as a “carrier” appears to take on the properties of that to the carrier in two significant respects: (i) 19s but no 7s antibody is detectable in the serum, as is also the case after injection of optimal doses of SIII alone; and (ii) immunological memory, that is, a higher response to a second injection than to the first, is absent as with SIII (23). This lack of immunological memory was seen both upon challenge of the intact “primed” mice and in adoptive immune responses. It was not due to an inhibition by serum antibody, since higher doses of the conjugates could not overcome this defect and, on the contrary, made the situation worse. The lack of memory was not only due to the shown inability of DNP-SIII to challenge 7s memory cells, but also to a lack of 7s memory formation after priming, since memory was equally lacking upon challenge of DNP-SIIIprimed cells with either DNP-BA or with DNP-SIII. Although the thymus-independent responses to polysaccharide antigens are frequently ascribed to the ability of these antigens to induce mitotic activity in B lymphocytes (3), the lack of memory formation characterizes the major
332
LERMAN
ET
AL.
deficiency of such responses and is probably due to insufficient proliferation of the B cells. Allogeneic effect (25, 26), antilymphocyte serum (27, 28)) and coupling to a thymus-dependent carrier (29, 30) may all result in significant increases of the response to SIII and other T-independent antigens in the mouse, in some cases accompanied by the appearance of 7S serum antibody (26, 28) and memory (31). It remains to be determined whether enhanced proliferation of the B lymphocytes, possibly in germinal centers, constitutes the major difference between the fullblown humoral immune response with resulting 19Sf7S antibody and memory, and the response to “thymus-independent” antigens lacking 7S antibody and memory. In nude, thymusless mice the response to SIII is normal or supranormal (27) as compared to littermate controls, even though all germinal center formation in nude mice is lacking. It should be noted, of course, that even in primary responses of nude and other mice to SIII a minor component of 7S antibodyforming cells does occur (31) .5 In the absence of T help the proliferation induced by polysaccharide antigens in B lymphocytes may drive all the antigen-sensitive precursors into terminal differentiation without establishment of a memory B-cell line, as suggested by recent studies of Howard and co-workers (32). The inability of TNP-SIII and DNP-SIII to challenge memory cells to TNPKLH was not simply due to the absence of interaction between the B memory cells and TNP-SIII. On the contrary, the hapten-SIII conjugates interacted so well with the TNP-memory cells that they blocked the response to the homologous antigen. This almost instantaneous abolition of responsiveness in TNP memory cells is in apparent contrast with the ability of these conjugates to induce primary responses to TNP. In view of the observed dose effect in the primary response, where 100 pg induced a lower response than did 1 pg while 1 PLg completely inhibited the secondary response to homologous antigen, a quantitative difference between primary and secondary responses suggests itself. It seems likely that such a difference between primary and memory responses is caused by the affinity of the cells for the antigen (TNP group) which is influenced by (i) the numbers of antibody molecules on the surface of the cell (IgM 7S monomers or 7S IgG molecules), and (ii) the avidity of these antibody molecules. It is well known that serum antibody attains a higher average avidity with time after immunization (33)) and it is less sure but likely that this property is shared by IgM and IgG (34, 35). Higher affinity of the cells for antigen would result in the concentration of much more antigen on their surface, resulting in a situation akin to the one seen with high conjugates of DNP-polymeric flagellin (36), or with antibody-antigen complexes (37, 38) which have been shown to facilitate induction of tolerance iti vitro, a tendency attributed to the need for a certain degree of lattice formation on the surface of the cells (39). It has also been suggested by Coutinho ef al. (24) that high concentrations of B-cell mitogens on the cell surface may in themselves be inhibitory rather than stimulatory for B-cell mitotic activity. The present results are consistent with (i) a higher avidity of both the IgM and IgG antibody on memory cells and (ii) a complete lack of proliferation and differentiation once the cells have interacted with too high a concentration of haptenSHI. Other explanations are also possible. ‘It is conceivable that a relative lack of 6 Similar findings have recently been obtained in chickens (M. becke, unpublished observations), in which low doses of DNP-SIII some mercaptoethanol-resistant serum antibody, but no memory.
D.
Grebenau and G. J. Thoror of SIII alone induced
BLOCK
OF
ANTI-HAPTEN
MEMORY
BY
HAPTEN-311
333
competition for antigen between B cells and macrophages in the primary response results in the stimulation of some B cells by antigen via a macrophage surface, whereas in the secondary response the higher affinity of the cells overrides such an intermediary role for macrophages and the direct interaction between the antigen and B cells causes tolerance. This implies the need for macrophages in thymus-independent responses, which so far has not been established in vitro (40). Tolerance in both unprimed and primed B cells has been induced in viva and in vitro by a variety of hapten conjugates to nonimmunogenic carriers (6, 11). Under special circumstances soluble hapten conjugates may interfere with the induction of a response in vitro by macrophage-bound hapten conjugates to the same immunogenic carrier (41). The avidity of the receptor on the B cell has been implicated in the sense that higher avidity always leads to a higher sensitivity to tolerance induction or to “blocking” (33, 42). In some such in z&o systems it was shown that several hours of incubation at 37°C were needed (43, 44). In the present study 5-15 min at 0°C sufficed, but further studies are required to determine whether or not a simple block of the receptors induced in vitro is followed by cellular changes at 37°C after transfer into the animal before unresponsiveness is established. Studies on the cellular mechanism and reversibility of this induction of tolerance in memory cells will be the subject of another publication (45). One striking similarity between induction of tolerance to hapten-polysaccharide conjugates and hapten conjugates to syngeneic substances is that they either do not involve T cells at all or are relatively inefficient at inducing T help via a new antigenie determinant (46). Nude mice which lack T cells can be rendered tolerant at the B-cell level with relative ease, even to thymus-dependent antigens (47). Fidler and Golub (48) also showed recently that free hapten (TNBS), which probably conjugates in the animals to produce TNP conjugates of syngeneic proteins, may induce tolerance in the absence of added T cells in B cells from fetal liver. The absence of suppressor activity in incubated, blocked cell populations is clearly shown in the present studies. It appears, therefore, that the absence of T help, rather than the presence of T suppressor cells, is crucial in the induction of tolerance in this system and that the sensitivity of B cells to such induction is dependent on the affinity of the cells for the antigen. It can be postulated that a high concentration of antigen directly bound to the B-cell surface induces tolerance or blocking, which is overcome if the cells are made to proliferate greatly by T help. Such a proliferation alone would be expected to dilute the antigen on the surface of B cells and cause a lowering of its concentration to immunogenic levels. This interpretation of tolerance induction at the B-cell level is supported by the recent suggestion of Benson and Bore1 (49) that tolerant cells are able to circulate with antigen bound to their surface. This might explain the continued block of receptors and interference with T help by carriers such as SIII which are nonimmunogenic for T cells. In view of recent observations that B cells form caps and strip their surface free of Ig within 1 hr after incubation with antigen (SO), an explanation is needed why blocked cells should keep the antigen on their surface for a time. Perhaps the multipoint surface interaction with antigen can initiate a state similar to the one described by Edelman and co-workers (51) after interaction of cells with tetramerit concanavalin A involving resistance to cap and patch formation by anti-Ig.
334
LERMAN
ET
AL.
Such changes on the cell surface might be reflected intracellularly by variations in mediator levels such as cyclic AMP, an increase which may cause inhibition of the immune response (5’2) and may be important as a signal for induction of tolerance (53). ACKNOWLEDGMENTS We are deeply indebted to Dr. A. Nisonoff, Department of Biochemistry, University Illinois Medical Center, for generous gifts of TNP-protein conjugates. We also wish thank Dr. C. E. Watson of the U.S. Department of Agriculture, Albany, NY for providing with the B. abortus used in these studies, In addition, we wish to express our appreciation Melvin Bell and Pedro Sanchez for their excellent technical assistance.
of to us to
REFERENCES 1. Feldman, M., and Basten, A., 2. Del Guercio, P., and Leuchars,
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
J. Exp. Med. 134, 103, 1971. E., J. Immunol. 109, 951, 1972. Coutinho, A., Gronowicz, E., Bullock, W. W., and Moller, G., J. Exp. Med. 139, 74, 1974. Schrader, J. W., J. Exp. Med. 137, 844, 1973. Del Guercio, P., Thobie, N., and Poirier, M. F., J. 1rnmzffl~o1. 112, 427, 1974. Katz, D. H., Davie, J. M., Paul, W. E., and Benacerraf, B., J. Exp. Med. 134, 201, 1971. Katz, D. H., Hamaoka, T., and Benacerraf, B., J. Exp. Med. 136, 1404, 1972. Nossal, G. J. V., Pike, B. I., and Katz, D. H., I. Exp. Med. 138, 312, 1973. Golan, D. T., and Borel, Y., J. Exp. Med. 134, 1046, 1971. Walters, C. S., Moorhead, J. W., and Claman, H. N., J. Exp. Med. 136, 56, 1972. Hamilton, J. A., and Miller, J. F. A. P., Eur. J. Immunol. 3, 457, 1973. Katz, D. H., Hamaoka, T., and Benacerraf, B., Proc. Nat. Acad. Sci. U.S.A. 70, 2776, 1973. Cohen, B. E., Davie, J. M., and Paul, W. E., J. Immzmol. 110, 213, 1973. Benacerraf, B., and Katz, D. H., J. Zmmmzol. 112, 1158, 1974. Mitchell, G. F., Humphrey, J. H., and Williamson, A. R., Eur. J. Immanol. 2, 460, 1972. Axen, R., and Ernback, S., Eur. J. Biochem. 18, 351, 1971. Rittenberg, M. B., and Pratt, K. L., Proc. Sot. Exp. Biol. Med. 132, 575, 1969. Little, J. R., and Eisen, H. N., ilz. “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), p. 128. Academic Press, New York, 1967. Heidelberger, M., Kendall, F. E., and Scherp, H. W., J. Exp. Med. 64, 559, 1936. Dubois, M., Giles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F., Anal. Gem. 28, 350, 1956. Sterzl, J., and Riha, I., Natitre (London) 208, 858, 1966. Stavitsky, A. B., J. Immnzmol. 72, 360, 1954. Baker, P. J., Stashak, P. W., Amsbaugh, D. F., and Prescott, B., Immt4woZogy 20, 469, 1971. Mond, J. J., Caporale, L., and Thorbecke, G. J., Cell. Immunol. 10, 105, 1974. Byfield, P., Christie, G. H., and Howard, J. G., J. Zmmunol. 111, 72, 1973. Ordal. 1. C.. and Grumet. F. C.. J. ExP. Med. 136, 1195, 1972. Baker: -P. J., Reed, N. ‘D., Stashak,. P. W., Amsbaugh, D. F., and Prescott, B., J. Exp. Med. 137, 1431, 1973. Barthold, D. R., Prescott, B., Stashak, P. W., Amsbaugh, D. F., and Baker, P. J., 1. Zmmunol. 112, 1042, 1974. Paul, W. E., Katz, D. H., and Benacerraf, B., J. Imrnz~rtnl. 107, 685, 1971. Byfield, P., Cell. Imnzunol. 3, 616, 1972. Paul, W. E., Banacerraf, B., Siskind, G. W., Goidl, E. H., and Reisfeld, K., J. Exp. Med. 130, 77, 1969. Howard, J. G., and Courtenay, B. M., Eur. J. Immunol. 4, 603, 1974. Siskind, G. W., and Benacerraf, B., in “Advances in Immunology” (F. J. Dixon, Jr, and H. G. Kunkel, Eds.), pp. l-50. Academic Press, New York, 1969. Claflin, L., and Merchant, B., Cell. Zmmunol. 5, 209, 1972. Wu, C-Y., and Cinader, B., Eur. J. Imnmnol. 2, 398, 1972. Feldman, M., Natllre New Biol. 231, 21, 1971.
BLOCK
37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.
OF
ANTI-HAPTEN
MEMORY
BY
HAPTEN-SIII
Feldman, M., and Diener, E., J. Exp. Med. 131, 247, 1970. Diener, E., and Feldman, M., J. Exp. Med. 132, 31, 1970. Diener, E., and Feldman, M., Cell. Immunol. 5, 130, 1972. Feldman, M., Eur. J. ImmunoZ. 2, 130, 1972. Katz, D. H., and Unanue, E. R., J. Exp. Med. 137, 967, 1973. Kettman, J., J. Iwnunol. 112, 1139, 1974. Diener, E., and Armstrong, W. D., J. Exp. Med. 129, 591, 1969. Britton, S., J. Exp. Med. 129, 469, 1969. Romano, T. J., Lerman, S. P., and Thorbecke. G. J., in preparation. Rubin, B., and Wigzell, H., J. Ex). Med. 137, 911, 1973. Schrader, J. W., J. Exp. Med. 139, 1393, 1974. Fidler, J. M., and Golub, E. S., J. Imm~4m1l. 112, 1891, 1974. Aldo-Benson, M., and Borel, Y., J. Iwwnu~zol. 112, 1793, 1974. Ault, K. A., and Unanue, E. R., J. Exp. Med. 139, 1110, 1974. Edelman, G. M., Yahara, I., and Wang, J. L., Proc. Nut. Acad. Bourne, H. R, Lichtenstein, L M., Melmon, K. L., Henry, Shearer, G. M., Science 184, 19, 1974. 53. Watson, J., Epstein, R., and Cohn, M., Nature 246, 405, 1973.
335
Sci. U.S.A. 70, 1442, 1973. C. S., Weinstein, Y., and
P.
LERMAN,
”
T. J.
KOMANO,
G. J. Department
of Pathology, New
New Fork,
a
J. J.
FROND,
&I.
HEIDELBERGER,
AND
THORBECXE York New
Unizlevsity School York 10016
of Medicble,
Trior dinitrophenylated pneumococcal polysaccharide type III (TNPor DNPSIII) ) induced a primary 19s anti-TNP response without generating immunological memory to the hapten in LAF, mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. a6ortus organisms (DNP-BA) induced both 19s and 7s primary responses and memory to the hapten. Spleen cells from mice immunized with TNPKLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLHprimed spleen cells for as short as 5 min at 0°C with 10 pg of TNP-SIII per milliliter virtually abolished their ability to give 19s and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.
INTRODUCTION Immunization with hapten coupled to a polysaccharide carrier which, by itself, induces thymus-independent immune responses, may give rise to anti-hapten antibody (1, 2). The mechanism of cellular interaction involved, if any, is unclear, but it has been suggested that direct mitogenic activity of these carriers for B cells is of prime importance in inducing T-cell independent responses (3, 4). The anti-hapten response may have similar properties as the response to its carrier in that it is limited to antibody production of the 19s class (1, 2) and deficient in the formation of immunological memory (5). On the other hand, hapten conjugates of nonimmunogenic molecules such as a copolymer of u-glutamic acid and D-lysine ( D-GL) (68)) syngeneic y-globulin (9, lo), and syngeneic red cells 1 Supported by Grant AI-3076 from the United States Public Grant QFB13592 AI from the National Science Foundation. 2 Supported by USPHS Training Grant No. GM000127. 3 Recipient of National Institutes of Health Training Grant National Institute of General Medical Science. 321 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Health
No.
Service
5GM01668-11
and
in part
by
from
the
322
LERMAN
ET
AL.
not appear to induce immm~e responses, but on the contrary may induce tolcrante to the hapten (11). It has been suggested that “no11iilllllllnoge~lic” hapten-carrier conjugates might have important applications in the abrogation of ongoing but mnwanted humoral responses (12). It would be of interest if hapten conjugates could be found which specifically abolish some but not all facets of the immune response to the hapten. For example, B-cell tolerogens such as the dinitrophenylated (DNP) D-GL inhibit antibody production while leaving intact T-cell functions with specificity for the hapten such as delayed and contact hypersensitivity (13, 14). Another promising approach to this problem was initiated by Mitchell et al. (15), who demonstrated interference with the 7s antibody memory response to DNP-hemocyanin through injection of the DNP-lysine conjugates of pneumococcal polysaccharide type III (DNP-SIII) . The present studies were undertaken to determine (i) whether the trinitrophenyl-lysine conjugates of SIII (TNP-SIII) could induce an immune response to TNP with generation of immunological memory, and (ii) whether or not the inhibition of 7S memory expression observed by Mitchell et al. (15) also encompassed 19s memory and could be obtained by in vitro incubation of memory cells.
do
MATERIALS
AND
METHODS
Animals and Immunizations Male young adult LAFl mice (Jackson Labs, Bar Harbor, ME) were used throughout. Donor mice were immunized by intravenous injection of 0.3 mg TNPla-KLH usually with 10 pg E. c&i endotoxin (Difco Laboratories, Inc., Detroit, MI). In a few experiments donor mice received 0.1 ml of a 0.3% (v/v) suspension (equivalent to 2 X 10R) of killed Brucelh abortus (BA) organisms. Whenever the interval prior to sacrifice of donor mice exceeded 2-4 months, they received a second dose intraperitoneally. Donors were used no earlier than 1 month after their last priming injection. Immunizations with TNP and DNP conjugates of SIII or of BA were always by intravenous injection of antigen in dosages as described in the tables. On the day prior to transfer, recipient mice received 630-750 R 13iCs v-irradiation from a gammator M (Radiation Machinery Corp., Parsippany, NJ ) . Antigens DNPSO-SIII and TNP,,,-SIII were prepared by conjugation to SIII of r-DNPL-lysine HCl (Sigma Chemical Co., St. Louis, MO) or e-TNP-L-lysine HCl (Nutritional Biochemicals Corp., Cleveland, OH), respectively, by the cyanogen halide method of Axen and Ernback (16) as described by Mitchell et al. (15). Trinitrophenylated sheep erythrocytes (TNP-SE) were prepared according to the method of Rittenberg and Pratt (17). TNP-BA and DNP-BA were prepared by the method of Little and Eisen (18). Briefly, trinitrobenzene-sulfonic acid (TNBS) was recrystallized from the commercial preparation (Eastman Kodak Co., Rochester, NY) by cooling from hot 1.0 N HCl and washing with cold HCl. To 20 mg of recrystallized TNBS or of dinitrobenzene-sulfonic acid (Eastman Kodak Co., Rochester, NY) dissolved in 2.0 ml of a 2% KzC03 solution were added 2.0 ml of packed BA (U.S. Department of Agriculture) with stirring. Stir-
BLOCK
OF
IZNTI-ITAPTEN
MEMORY
lo2o-7
RY
11 .‘iPTEN-SIII
323
6 Days
After
Injection
FIG. 1. Primary anti-TNP responses of LAF, mice expressed as PFC/spleen at various days and TNP-B.4 (A-A) or 1 ,~g (O-0) and after injection of DNP-BA (n---A) 100 pg (O--O ) DNP-SIII. Each point represents the mean f SE for 2-9 mice. Shaded area denotes the mean PFC level for normal spleen 5 SE measured with TNP-sheep erythrocytes. The numbers of PFC/spleen against sheep erythrocytes were subtracted from all values.
ring was continued for an additional 2 hr at room temperature and overnight at 0°C. The mixture was dialyzed successively against distilled water, 0.1 M KC2H302, and finally 0.15 &f NaCl. Dinitrophenyl groups were conjugated to human gamma globulin (HGG) and keyhole limpet hemocyanin ( KLH) (Mann Laboratories, Orangeburg, NY) (18). The extent of coupling of DNP to HGG, KLH, and SIII was estimated from the molar extinction coefficient, 17,400, at 360 nm with 100,000 as the unit of molecular weight for the protein and polysaccharide carriers (15). The degree of TNP conjugation was estimated using the molar extinction coefficient, 15,400, at 346 nm. SIII was prepared as described previously (19), and the amount of polysaccharide present in each of our hapten-polysaccharide conjugates was determined by the phenol-sulfuric acid reaction of Dubois et a.1. (20). Cell Transfers
The spleens of donor mice were teased into a cell suspension in Medium 199 (Microbiological Associates Inc., Bethesda, MD) and washed twice. Cells were either injected intravenously into irradiated recipients with antigen or were incubated with antigen in zitro prior to transfer as described under Results. Assays
Numbers of antibody-forming cells per spleen to TNP were enumerated by localized hemolysis in gel of TNP-SE (17). A s a control, spleen cell suspensions were also plated against uncoated SE and the values obtained subtracted from those for PFC against TNP-SE. Rabbit anti-mouse Ig was used for development of indirect PFC (21). The numbers of 7S PFC were calculated by subtracting the direct PFC from the indirect PFC. Sera from recipient mice were titrated for anti-TNP and anti-KLH by passive hemagglutination. Indicator cells were tanned cells (22) to which had been adsorbed 0.05 mg TNP1.7-ovalbumin or KLH per milliliter of 2.5% tanned SE.
324
LERMAN
ET
i\L.
RESULTS Primary
Response
to Hapten
Presented
011 the BA and SIII
Carriers
Initial studies of responses to a single injection of DNl’-]:A, TNP-BA, DNl’SIII, and TNP-ST11 showed that all induced a rise in the number of PFC per spleen with anti-TNP specificity, but with large differences in effectiveness between the SIII and the BA carrier. Figure 1 shows the responses to DNP-BA and TNP-BA. Peak PFC/spleen values were reached on day 3, and the number of antibody-forming cells remained high for several days. TNP-BA always gave a much higher response than three different preparations of DNP-BA, and at the dose used was also more effective than either TNP-KLH or DNP-KLH given as a single intravenous dose of 100 pg. The mean PFC/spleen on day S after TNP-KLH was 4175 as compared to 583 for DNP-KLH. 4 The high immunogenicity of BA conjugates might be due to their probable content of endotoxin. Indeed, immunization with DNP-KLH, given together with 50 pg of E. coli endotoxin, resulted in a response at least as high as that to DNP-BA. Although the majority of the PFC detected through day 6 after injection of KLH or BA conjugates were 19S-producing, a significant percentage of animals also showed 7S antibody-producing cells on day 6 of the primary response. The response to DNPor TNP-SIII, on the contrary, did not show a significant 7s component at any time. Peak PFC/spleen values after an optimal (1 pg) dose of DNP-SIII were reached on day 3 and declined thereafter (Fig. 1). Ten micrograms of DNP-SIII induced a similar response, but that to 100 pg was much lower (Fig. 1) . Control LAFl mice injected with 0.1 ml of 10m5or of 1c6 M TNP-lysine containing 1 pg unconjugated SIII did not show any splenic PFC above background 4 days later. The response to TNP-SIII was slightly higher than to DNP-SIII but otherwise was similar. Mean PFC/spleen values on days 4-5 were 3550 after 1 pg, 3218 after 10 pg, and 724 after 100 pg of TNP-SIII. Experiments in which immunization with DNP-SIII was attempted 1 week after a previous tolerogenic or immunogenic dose of SIII were difficult to interpret, since both dose levels of SIII used appeared to lower somewhat the subsequent response to DNP-SIII. Secondary
Response
in Yivo
to Haptens
Presented
on BA
and SIII
Carriers
Mice reinjected with 1 pg DNP-SIII 3 weeks after a primary injection of 1, 10, or 100 pg DNP-SIII did not show a secondary response. Direct PFC/spleen values measured 6 days later were no different from those in unchallenged mice and were similar to those seen 5 days after a primary injection (Expt 1, Table 1). 7s PFC were absent. DNP-BA induced a slightly higher response comparable to the one seen on day 6 after a primary injection of this antigen. There was no 4In this experiment all PFC determinations were made against TNP-SE rather than DNP-SE. That higher PFC values after immunization with TNP as compared to DNP conjugates were not due to the method of detection used was shown in unpublished observations with Dr. E. Goidl and Dr. G. W. Siskind, Department of Medicine, Cornell University Medical Center, New York, in which PFC detected with DNP-SE as indicator cells were also more numerous after immunization with TNP conjugates. Thus, TNP conjugates appear to have higher immunogenicity for LAFl mice than do DNP conjugates.
BLOCK
OF
ANTI-IIAPTEN
MEMORY
BY
IIAl’TEN-SIII
~MhlliNOl.OGlCAI,
h!EMOR\'
TABLE INABILITY
OF UNP-SIII
TO Imuce
1
Challengmg antigen
Priming antigen
Mean
Expt
Direct (1 ,ug)
Expt
1”
3.25
f 0.09 (1760)
3.39
f 0.15 (2425)
DNP-BA
3.52
z!c 0.10 (3290)
3.53
f 0.17 (3405)
Direct
3.35
TNP-BA
DNP-SIII
(10 fig)
3.18~ (1510) 3.36
f 0.07 (2270)
3.19
i 0.13 (155.5)
DNP-BA
3.66
f 0.10 (4535)
3.67
zk 0.12 (4670)
TNP-BA
DNP-SIII
(100
rg)
DNP-BA
A 0.13 (2255)
3.36 f 0.16 (2270)
4.45 f 0.07 (28,150)
4.65 f 0.13 (44,500)
3.30
3.29
f 0.12 (2010)
4.38 =!z 0.25 (24,200) 3.18~ (1520) 3.19
f 0.12 (1555)
3.23
i 0.08 (1710)
DNP-BA
3.45
f 0.07 (2785)
3.46
zk 0.06 (2850)
3.1Sc (1510) 3.58
41 0.06 (3830)
3.64
DNP-BA
3.69
f 0.08 (4930)
4.06 zt 0.09 (11,450)
3.14c (1370)
4.S6 f 0.22 (36.480)
3.16< (1460)
DNP-SIII
None
zk 0.07 (1940)
3.20c (1.570)
DNP-SIII
None
Indirect
3.11c (1300)
DNP-SIII
None
2”
__~
Indirect
DNP-SIIl*
None
TO DNP
log PFC/spleen & SE (geometric mean)
.___..~
DNP-SIII
32.5
zt 0.06 (4410’
3.3.V (2230)
a Interval between injections was 21 days (Expt 1) or 14 days each group varied from 4 (Expt 1) to 2 or 3 (Expt 2). Determination day 6 (Expt 1) or day 5 (Expt 2) after challenge. b Challenge dose was 1 pg DNP-SIII in all experiments. c Recipients’ spleens in these groups were pooled prior to assay
(Expt 2). Numbers of PFC/spleen
for
of mice in was done on
PFC.
tolerance to DNP in mice previously injected with 100 pg DNP-SIII since they were still able to respond to DNP-BA, although possibly somewhat less than normal mice. In Expt 2, mice reinjected with DNP-BA and TNP-BA 2 weeks after priming with DNP-911 gave similar results. Responses comparable to those
326
LERMAN
ET
TABLE
Donors primed with
AL.
2
Recipients challenged with
-VI/lean log PFC/spIeen (geometric mean)
2.65
9’
Expt
Indirect
Direct DNP-BA
AI 0.05
3.68
(442) DNP-SIII”
1.76
f
0.45
1.81
Direct
f 0.09 (4810) f
0.18
DNP-KLH*
DNP-SIII
(1 ,.‘g)
(100 pg)
2.36
f 0.16 (229)
2.26
f
0.08
(182)
DNP-BA
3.28
zk 0.13 (1905)
3.21
f 0.06 (1620)
DNP-SIII
3.19
f 0.19 (1550)
3.05
zt 0.09 (1130)
DNP-BA DNP-SIII None
2.75
3.2@ (1840) 3.22
f 0.08 (1660)
2.85
f 0.21 (709)
3.08c (1200)
Indirect
zt 0.04 (1290)
3.70
rt 0.14
2.68
f 0.13 (1160)
.-.
@ -___
(563) 3.07
None
DNP-SIII
3.11
(65)
(57)
None
SE ____-.
Expt
DNP-BA
f
f 0.08 (5200) f
0.18
(479) 3.11
f 0.14 (1305)
2.93c
2.94c
(851)
(870)
3.08c (1190) 2.87
f
0.18
(742) 2.79
f 0.14 (617) ND”
a Interval between priming and transfer was 16 weeks for the DNP-BA-primed spleen cells and 4 weeks: for DNP-SIII-primed cells in Expt 5, and 3 weeks for DNP-BA-primed spleen cells in Expt 6. Irradiated recipients received 3-3.5 X lo7 cells (Expt 5) or 2 X lo7 cells (Expt 6) together with the challenging antigen. Receipients’ spleens were assayed for PFC 7 days later (4-5 mice per group). * All challenges with DNP-SIII employed 1 rg DNP-SIII except for Expt 5 (donors primed with DNP-BA), where the dose was 50 pg DNP-SIII. Dose of DNP-KLH was 100 rg. c Recipients’ spleens in these groups were pooled prior to assay for PFC. * Not determined.
after a primary injection of these antigens were found on day 5 after challenge, and a 7s component was detectable as also seen after the primary injection. In contrast, mice primed with DNP-BA gave a secondary 7s anamnestic response to DNP which was not demonstrable after challenge with DNP-SIII. There was a slightly higher 19s response to DNP-SIII in these mice than in those primed with DNP-SIII. Adoptive
Secondary
Responses
to DNP-BA
and DNP-SIII
Since it was conceivable that the presence of antibody to the SIII carrier molecule in the sera of primed mice was inhibiting the secondary response to
BLOCK
OF
ANTI-HAPTEN
MEMORY ‘TARI,E
~\RII.ITY
OF DW-I%\
Recipients challenged
AKD
BY
3
DNP-HGG TO ELICIT AN XDOPTIVE DNP-KLH-PKIMED SPI.EIX CELLS hlean
with
327
HAPTEN-SIII
Smmna~r
RESPONSE
log PFC/spleen =t SE (geometric mean) Expt
Expt 31’ Direct Direct
+ Indirect
DNPzG-KLH*
3.21
f 0.08 (1623)
3.38
f 0.09 (2380)
4.40 * 0.04 (25,200)
DNP-RA
3.12
f 0.04 (1320)
3.26
f 0.09 (1830)
3.81
f 0.22 (6460)
DNPj-HGG
2.92
0.05
3.28
f 0.04 (1895)
3.70
f 0.04 (5020)
0.43
3.01
f 0.21 (1025)
3.20
f 0.08 (1585)
f (832)
DNPtg-HGG
1.26
f
(18) n Spleen cells were taken 2 weeks (Expt 3) or 3 KLH with or without 10 rg E. coli endotoxin. 2 X 107 (Expt 4) spleen cells together with the assayed for PFC 6 days (Expt 3) or 7 days (Expt b Challenge dose of antigen, 0.2 mg DNP-KLH
FKOY
weeks {(Expt 4) after injection of 1 mg DNPZBIrradiated recipients received 10’ (Expt 3) or challenging antigens. Recipients’ spleens were 4) later (4-5 mice per group). or 0.1 mg DNP-HGG.
these conjugates, adoptive secondary responses after transfer of 2-3 x lo7 spleen cells from immunized mice were studied. Table 2 shows the results of these experiments. Cells taken 4 weeks after injection of DNP-SIII did not respond to either DNP-BA or DNP-SIII, giving similar PFC values in recipient spleens with or without antigenic challenge at the time of transfer. DNP-BA-primed spleen cells showed definite 7s and weak 19s anamnestic responses to DNP-BA but none to DNP-SIII. These data implied that DNP-SIII was unable to generate memory cells to DNP and, thus, behaved like SIII alone, which is known to induce 19s responses without generating detectable memory (22). However, the possibility existed that DNP-SIII had generated memory cells but was unable to challenge them and that DNP-BA could not challenge this memory to DNP-SIII because of the difference in carrier. Although BA itself is a largely thymus-independent antigen with respect to challenge of memory cells (23), the DNP-BA conjugate might not be equally thymus independent. We therefore tested the capacity of DNP-BA to challenge memory cells generated by DNP-KLH. Spleen cells from DNP-KLHprimed mice were taken 2 weeks (Expt 3, Table 3) or 3 weeks (Expt 4, Table 3) after immunization and challenged up011 transfer (2 x 10’ cells/mouse) with DNPKLH, DNP-BA, or DNP-HGG conjugates. DNP-BA was much more efficient than DNP-HGG in challenging these memory cells, although it induced a lower 7S response than the homologous DNP-KLH conjugate. It should, therefore, have been capable of challenging memory cells to DNP-SIII if such cells had been present.
328
LERMAN
ET
AL.
The previous experiments showed that DNP-SIII neither generated nor challenged DNP memory cells. We next examined the ability of these conjugates to b10clc the responsiveness of memory cells in V~ZIO.Table 4 shows that, when haptenHA- or hapten-KLH-prinie~l memory cells were transferred with the homologous antigen and varying doses of the hapten-SIII conjugates, both 19s and 7S nienlory cells did not respond to the homologous conjugate. Even as little as 0.5-l pg of hapten-SIII resulted in significant inhibition of both responses. In two of the experiments in which this comparison could be made the 19s responses appeared slightly less sensitive to inhibition than the 7s responses (Expts 11 and 12, Table 4). When passive hemagglutination titers to TNP of the recipients’ sera were compared with values for PFC/spleen, agreement between these two measures of immune responsiveness of the transferred cells was noted. In experiments in which the PFC/spleen values indicated an effective block of the response, the anti-TNP titers of the sera were also greatly reduced, while a less effective block of the PFC/spleen response resulted in a smaller reduction in titer. For example, in three typical experiments, the control recipient groups had mean log2 serum titers ranging from 10.0 to 10.8, the groups which did not receive antigen had mean log, titers of 2.0-2.7, and the mice receiving TNP-KLH with 5-50 pg TNP-SIII had mean log2 titers of 2.3-3.3. Mice receiving a lower dose of the blocking antigen (0.5-l pg) had titers of 7.4-8.7. Further studies showed that a simple preincubation with hapten-SIII for as short as 5 min at O”C, followed by washing of the cells and transfer into irradiated recipients together with the homologous conjugate, was sufficient to block partially the response of TNP memory cells (Table 5). Excellent correlation was again found between results obtained for PFC per spleen and for anti-TNP titers in recipients’ sera (Table 6). Incubation at 0°C was no less effective than at 37”C, and prolonging the incubation to 60 min did not increase the degree of inhibition (Table 5)) nor did increasing the concentration of DSP-SIII to 200 rg/ml (45% vs 417*). Although the results were somewhat variable, TKP-SIII was more effective than DNP-SIII, particularly at the 10 pg/ml level, possibly because of the much higher degree of conjugation of ST11 with TNP than with DNP. UThile only one experiment was included in Table 5, good inhibition was obtained at this level of TNP-SIII in several additional experiments. It was noted that both 19s and 7S memory cells were sensitive to the blocking effect of preincubation. Preincubation with 40 pg DNP-SIII/ml, followed by washing, was slightly less effective in blocking the subsequent response to TNP-KLH than the simultaneous injection of 0.5 PLgof DNP-SIII (Expt 12, Tables 4 and 5). Additional experiments (not in the tables) showed that incubation of TNPKLH primed cells with 100 pg/ml of either DNPB-HGG or DNPhS-HGG, followed by washing had no detectable blocking effect on the subsequent adoptive response to TNP-KLH. Controls
for Specificity
of the phenomenon was established by an experiment in which cells to burro erythrocytes were exposed for 1 hr at 0°C to 40 pg TNP-SIII/ml.
Specificity primed
and Suppressor Eflects
S-50 0.5 1 l-50 None
+ + -
100 (1660) 4 20 10 1
19s
Expt
8 7s
ANTIGEN
100 (1790) 3 37
TO HOMOLOGOUS
4
7s
9=
100 (1339) 3
Expt
REMAINING
TABLE
7s
10~ 19s
in Expts were
and antigen
16
53
Expt
16
100 (14,904)
7s
not
significantly
above
Expt
7s
for
(0.1
2
4
100 (35,329)
12
the values
TNP-KLH
19
41
100 (2891)
19s
12 it was
ANTIGEN
9, 11, and
11
OF BLOCKING
100 (16,932)
IXJECTION
(PFC/spleen)
100 (1501)
Expt
7.
AFTER
in Expts 7 and 10 was DNP-BA, in Expt 8 it was TNP-BA, lo-12 was DNP-SIII, and in Expts 8 and 9 it was TNP-SIII. are not included because the 19s responses to homologous
3
(6371
100
Expt 7~ 7s
OF RESPONSE
a The homologous challenging antigen * The blocking antigen in Expts 7 and c In these experiments the 19s values injected without antigen.
None
Blocking antigen6 kd
+
Homologous antigen challenge5
PERCENT
cells
mg).
I I (40’
(10) (40) (10-20) onlyb
TNP-SI
TNP-SIII DNP-SIII DNP-SIII Medium
60 5 5 60 60 60
60
Incubation time (min)
34 16
7s
83 <1
100 (14,904)
11
RESPOXSE
Expt
100 (16,932)
19s
OF SECONDARY
Expt
19
28
100 (2891)
19s
TO TNP-KLH
7S
2
9 18
100 (3289) 75 23
19s
Expt
5
100 (26,389) 23 8
7S
13
‘% (PFC/spleen)”
INCUBATION
11
100 (3529) 3.5
19s
Expt
OF PRIMED
or TNP-SIII at O’C as indicated. They were determined 7 days after transfer.
100 (35,329)
12
REMAININGAFTER
D Cells were preincubated at 2 X 10’ cells/ml with or without DNPcells per recipient with 0.1 mg TNP-KLH. PFC/spleen in recipients b Control cells, not challenged with TNP-KLH after transfer.
only
incubated with CudmU
Medium
Cells
PERCENT
TABLE
75
were
19s
twice
3
12
Expt
DNP-
100 (9671)
WITH
washed
100 (28,321) 3
14
CELLS
7S
prior
19s
40 52 4 of l-2
Expt
100 (1356)
to injection
18
100 (13,922)
15
ORTNP-SIII
7s
X lo7
61 81
100 (1902)
16
F
3
2i
E
E
BLOCK
INHIBITORY
EFFECT
OF
Expt
Medium
only
Medium
Geometric mean PFC/spleen
TNP-KLH Medium
SPLEEN
ANTI-TNP
RUT
KOT
ON
CELLS Expt
14
17
~ Mean logz hemagglutination titer
31,850
TNP-KLH only
331
HAPTEN-SIII
ON ADOPTIVE
OF TNP-KLH-PRIMED
antiTNP
TNP-SIII
BY
WITH
RESPONSES
Cells injected with
MEMORY
TABLE 6 TNP-SIII
OF PKEINCUBATION
ANTI-KLH Cells incubated witha
ANTI-HAPTEN
only
10.8
1,514 606
a Cells were preincubated at 2 X lo7 cells/ml at 0°C. They were washed twice prior to injection per recipient with 0.1 mg TNP-KLH. PFC/spleen mined 7 days after transfer.
2.8 2.7
Geometric mean PFC/spleen
antiKLH 13.3
14.3 7.0
Mean log, hemagglutination titer antiTNP
25,365 4,562 6i7
9.6 4.8 4.0
antiKLH
15.6 7.0 17.7
with or without 40 pg/ml TNP-SIII for 60 min of 1 X 107 (Expt 14) or 2 X lo7 (Expt 17) cells vs TNP and serum antibody titers were deter-
The adoptive memory response of these cells, approximately 6000 7s PFC/spleen, was totally unaffected. Furthermore, while serum titers to TNP of recipients were reduced in parallel with numbers of PFC per spleen, the preincubation of donor cells with the blocking antigen had no detectable effect on the response to KLH, the carrier protein, as determined by serum antibody titers (Table 6). Also, transfer of a combination of unincubated TNP-KLH memory cells with an equal number of such cells after incubation with TNP-SIII and washing gave similar responses to those of unincubated cells showing both the absence of suppressor activity and of excess TNP-911 with the incubated cells. DISCUSSION The results indicate that DNP-SIII, like DNP-levan (2), induces a primary response to DNP in mice which is dose dependent in the sense that high doses (100 pg) give a lower response than low doses (0.5 pg). The response to the hapten on the SIII molecule as a “carrier” appears to take on the properties of that to the carrier in two significant respects: (i) 19s but no 7s antibody is detectable in the serum, as is also the case after injection of optimal doses of SIII alone; and (ii) immunological memory, that is, a higher response to a second injection than to the first, is absent as with SIII (23). This lack of immunological memory was seen both upon challenge of the intact “primed” mice and in adoptive immune responses. It was not due to an inhibition by serum antibody, since higher doses of the conjugates could not overcome this defect and, on the contrary, made the situation worse. The lack of memory was not only due to the shown inability of DNP-SIII to challenge 7s memory cells, but also to a lack of 7s memory formation after priming, since memory was equally lacking upon challenge of DNP-SIIIprimed cells with either DNP-BA or with DNP-SIII. Although the thymus-independent responses to polysaccharide antigens are frequently ascribed to the ability of these antigens to induce mitotic activity in B lymphocytes (3), the lack of memory formation characterizes the major
332
LERMAN
ET
AL.
deficiency of such responses and is probably due to insufficient proliferation of the B cells. Allogeneic effect (25, 26), antilymphocyte serum (27, 28)) and coupling to a thymus-dependent carrier (29, 30) may all result in significant increases of the response to SIII and other T-independent antigens in the mouse, in some cases accompanied by the appearance of 7S serum antibody (26, 28) and memory (31). It remains to be determined whether enhanced proliferation of the B lymphocytes, possibly in germinal centers, constitutes the major difference between the fullblown humoral immune response with resulting 19Sf7S antibody and memory, and the response to “thymus-independent” antigens lacking 7S antibody and memory. In nude, thymusless mice the response to SIII is normal or supranormal (27) as compared to littermate controls, even though all germinal center formation in nude mice is lacking. It should be noted, of course, that even in primary responses of nude and other mice to SIII a minor component of 7S antibodyforming cells does occur (31) .5 In the absence of T help the proliferation induced by polysaccharide antigens in B lymphocytes may drive all the antigen-sensitive precursors into terminal differentiation without establishment of a memory B-cell line, as suggested by recent studies of Howard and co-workers (32). The inability of TNP-SIII and DNP-SIII to challenge memory cells to TNPKLH was not simply due to the absence of interaction between the B memory cells and TNP-SIII. On the contrary, the hapten-SIII conjugates interacted so well with the TNP-memory cells that they blocked the response to the homologous antigen. This almost instantaneous abolition of responsiveness in TNP memory cells is in apparent contrast with the ability of these conjugates to induce primary responses to TNP. In view of the observed dose effect in the primary response, where 100 pg induced a lower response than did 1 pg while 1 PLg completely inhibited the secondary response to homologous antigen, a quantitative difference between primary and secondary responses suggests itself. It seems likely that such a difference between primary and memory responses is caused by the affinity of the cells for the antigen (TNP group) which is influenced by (i) the numbers of antibody molecules on the surface of the cell (IgM 7S monomers or 7S IgG molecules), and (ii) the avidity of these antibody molecules. It is well known that serum antibody attains a higher average avidity with time after immunization (33)) and it is less sure but likely that this property is shared by IgM and IgG (34, 35). Higher affinity of the cells for antigen would result in the concentration of much more antigen on their surface, resulting in a situation akin to the one seen with high conjugates of DNP-polymeric flagellin (36), or with antibody-antigen complexes (37, 38) which have been shown to facilitate induction of tolerance iti vitro, a tendency attributed to the need for a certain degree of lattice formation on the surface of the cells (39). It has also been suggested by Coutinho ef al. (24) that high concentrations of B-cell mitogens on the cell surface may in themselves be inhibitory rather than stimulatory for B-cell mitotic activity. The present results are consistent with (i) a higher avidity of both the IgM and IgG antibody on memory cells and (ii) a complete lack of proliferation and differentiation once the cells have interacted with too high a concentration of haptenSHI. Other explanations are also possible. ‘It is conceivable that a relative lack of 6 Similar findings have recently been obtained in chickens (M. becke, unpublished observations), in which low doses of DNP-SIII some mercaptoethanol-resistant serum antibody, but no memory.
D.
Grebenau and G. J. Thoror of SIII alone induced
BLOCK
OF
ANTI-HAPTEN
MEMORY
BY
HAPTEN-311
333
competition for antigen between B cells and macrophages in the primary response results in the stimulation of some B cells by antigen via a macrophage surface, whereas in the secondary response the higher affinity of the cells overrides such an intermediary role for macrophages and the direct interaction between the antigen and B cells causes tolerance. This implies the need for macrophages in thymus-independent responses, which so far has not been established in vitro (40). Tolerance in both unprimed and primed B cells has been induced in viva and in vitro by a variety of hapten conjugates to nonimmunogenic carriers (6, 11). Under special circumstances soluble hapten conjugates may interfere with the induction of a response in vitro by macrophage-bound hapten conjugates to the same immunogenic carrier (41). The avidity of the receptor on the B cell has been implicated in the sense that higher avidity always leads to a higher sensitivity to tolerance induction or to “blocking” (33, 42). In some such in z&o systems it was shown that several hours of incubation at 37°C were needed (43, 44). In the present study 5-15 min at 0°C sufficed, but further studies are required to determine whether or not a simple block of the receptors induced in vitro is followed by cellular changes at 37°C after transfer into the animal before unresponsiveness is established. Studies on the cellular mechanism and reversibility of this induction of tolerance in memory cells will be the subject of another publication (45). One striking similarity between induction of tolerance to hapten-polysaccharide conjugates and hapten conjugates to syngeneic substances is that they either do not involve T cells at all or are relatively inefficient at inducing T help via a new antigenie determinant (46). Nude mice which lack T cells can be rendered tolerant at the B-cell level with relative ease, even to thymus-dependent antigens (47). Fidler and Golub (48) also showed recently that free hapten (TNBS), which probably conjugates in the animals to produce TNP conjugates of syngeneic proteins, may induce tolerance in the absence of added T cells in B cells from fetal liver. The absence of suppressor activity in incubated, blocked cell populations is clearly shown in the present studies. It appears, therefore, that the absence of T help, rather than the presence of T suppressor cells, is crucial in the induction of tolerance in this system and that the sensitivity of B cells to such induction is dependent on the affinity of the cells for the antigen. It can be postulated that a high concentration of antigen directly bound to the B-cell surface induces tolerance or blocking, which is overcome if the cells are made to proliferate greatly by T help. Such a proliferation alone would be expected to dilute the antigen on the surface of B cells and cause a lowering of its concentration to immunogenic levels. This interpretation of tolerance induction at the B-cell level is supported by the recent suggestion of Benson and Bore1 (49) that tolerant cells are able to circulate with antigen bound to their surface. This might explain the continued block of receptors and interference with T help by carriers such as SIII which are nonimmunogenic for T cells. In view of recent observations that B cells form caps and strip their surface free of Ig within 1 hr after incubation with antigen (SO), an explanation is needed why blocked cells should keep the antigen on their surface for a time. Perhaps the multipoint surface interaction with antigen can initiate a state similar to the one described by Edelman and co-workers (51) after interaction of cells with tetramerit concanavalin A involving resistance to cap and patch formation by anti-Ig.
334
LERMAN
ET
AL.
Such changes on the cell surface might be reflected intracellularly by variations in mediator levels such as cyclic AMP, an increase which may cause inhibition of the immune response (5’2) and may be important as a signal for induction of tolerance (53). ACKNOWLEDGMENTS We are deeply indebted to Dr. A. Nisonoff, Department of Biochemistry, University Illinois Medical Center, for generous gifts of TNP-protein conjugates. We also wish thank Dr. C. E. Watson of the U.S. Department of Agriculture, Albany, NY for providing with the B. abortus used in these studies, In addition, we wish to express our appreciation Melvin Bell and Pedro Sanchez for their excellent technical assistance.
of to us to
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BY
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335
Sci. U.S.A. 70, 1442, 1973. C. S., Weinstein, Y., and